The present invention relates generally to fluorescent dye compounds that are useful as molecular probes. In particular, the present invention relates to fluorescent rhodamine dye compounds that are photostable and highly water-soluble.
The non-radioactive detection of nucleic acids utilizing fluorescent labels is an important technology in modem molecular biology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact and costs associated with reagent disposal is greatly reduced. Examples of methods utilizing such nonradioactive fluorescent detection include automated DNA sequencing, oligonucleotide hybridization methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
In many applications it is advantageous to employ multiple spectrally distinguishable fluorescent labels in order to achieve independent detection of a plurality of spatially overlapping analytes, i.e., multiplex fluorescent detection. Examples of methods utilizing multiplex fluorescent detection include single-tube multiplex DNA probe assays, PCR, single nucleotide polymorphisms and multi-color automated DNA sequencing. The number of reaction vessels may be reduced thereby simplifying experimental protocols and facilitating the production of application-specific reagent kits. In the case of multi-color automated DNA sequencing, multiplex fluorescent detection allows for the analysis of multiple nucleotide bases in a single electrophoresis lane thereby increasing throughput over single-color methods and reducing uncertainties associated with inter-lane electrophoretic mobility variations.
Assembling a set of multiple spectrally distinguishable fluorescent labels useful for multiplex fluorescent detection is problematic. Multiplex fluorescent detection imposes at least six severe constraints on the selection of component fluorescent labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and/or treatment with enzymes, e.g., automated DNA sequencing. First, it is difficult to find a set of structurally similar dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm). Second, even if dyes with non-overlapping emission spectra are identified, the set may still not be suitable if the respective fluorescent quantum efficiencies are too low. Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are usually widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the analyte. Fifth, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the analyte, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like. Sixth, the dye must have sufficient photostability to withstand laser excitation.
Currently available multiple dye sets suitable for use in four-color automated DNA sequencing applications require blue or blue-green laser light to adequately excite fluorescence emissions from all of the dyes making up the set, e.g., argon-ion lasers. As lower cost red lasers become available, a need develops for fluorescent dye compounds and their nucleic acid conjugates which satisfy the above constraints and are excitable by laser light having a wavelength above about 500 nm.
These and other objects are furnished by the present invention, which in one aspect provides water-soluble, photostable rhodamine dye compounds that can be used as labels in a variety of biological and non-biological assays. Generally, the rhodamine dye compounds of the invention comprise a rhodamine-type parent xanthene ring substituted at the xanthene C-9 carbon with a substituted phenyl ring. The substituted phenyl ring contains three to five substituents including: an ortho carboxyl or sulfonate group; one or more aminopyridinium (xe2x80x9cPyr+xe2x80x9d) groups; and one alkylthio, arylthio or heteroarylthio group. The alkylthio, arylthio or heteroarylthio group is believed to be positioned para to the carboxyl or sulfonate group, with the remaining positions being substituted with Pyr+ groups.
The aminopyridinium groups are attached to the phenyl ring at the pyridinium ring nitrogen and may be substituted or unsubstituted at the pyridinium ring carbons with one or more of a wide variety of the same or different substituents. The substituents may be virtually any group. However, electron-withdrawing groups (e.g., xe2x80x94NO2, xe2x80x94F, xe2x80x94Cl, xe2x80x94CN, xe2x80x94CF3, etc.) should not be attached directly to the pyridinium ring carbons, as these substituents may adversely affect the synthesis of the rhodamine dyes. Electron-withdrawing groups may be included on a substituent as long as it is spaced away from the pyridinium ring so as to not adversely affect the synthesis of the dyes. Thus, typical pyridinium ring carbon substituents include, but are not limited to xe2x80x94R, xe2x80x94OR, xe2x80x94SR, xe2x80x94NRR, xe2x80x94S(O)2Oxe2x80x94, xe2x80x94S(O)2OH, xe2x80x94S(O)2R, xe2x80x94C(O)R, xe2x80x94C(O)X, xe2x80x94C(S)R, xe2x80x94C(S)X, xe2x80x94C(O)OR, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)OR, xe2x80x94C(O)SR, xe2x80x94C(S)SR, xe2x80x94C(O)NRR, xe2x80x94C(S)NRR and xe2x80x94C(NR)NRR, where each R is independently hydrogen, (C1-C6) alkyl, or heteroalkyl, (C5-C14) aryl or heteroaryl. The R groups may be further substituted with one or more of the same or different substituents, which are typically selected from the group consisting of xe2x80x94X, xe2x80x94Rxe2x80x2, xe2x95x90O, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x95x90S, xe2x80x94NRxe2x80x2Rxe2x80x2, xe2x95x90NRxe2x80x2, xe2x80x94CX3, xe2x80x94CN, xe2x80x94OCN, xe2x80x94SCN, xe2x80x94NCO, xe2x80x94NCS, xe2x80x94NO, xe2x80x94NO2, xe2x95x90N2, xe2x80x94N3, xe2x80x94S(O)2O, xe2x80x94S(O)2OH, xe2x80x94S(O)2Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94C(O)X, xe2x80x94C(S)Rxe2x80x2, xe2x80x94C(S)X, xe2x80x94C(O)ORxe2x80x2, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)ORxe2x80x2, xe2x80x94C(O)SRxe2x80x2, xe2x80x94C(S)SRxe2x80x2, xe2x80x94C(O)NRxe2x80x2Rxe2x80x2, xe2x80x94C(S)NRxe2x80x2Rxe2x80x2 and xe2x80x94C(NR)NRxe2x80x2Rxe2x80x2, where each X is independently a halogen (preferably xe2x80x94F or xe2x80x94Cl) and each Rxe2x80x2 is independently hydrogen, (C1-C6) alkyl or heteroalkyl, (C5-C14) aryl or heteroaryl. Preferably, the pyridinium ring carbons are unsubstituted. When substituted, the most preferred substituents are the same or different (C1-C6) alkyls.
The amino group of the aminopyridinium groups is located at the 4-position of the pyridinium ring. The amino group may be a primary, secondary or tertiary amino group, but is typically a tertiary amino. The nitrogen substituents are typically (C1-C6) alkyl groups or heteroalkyl groups, and may be the same or different. Alternatively, the nitrogen is substituted with an alkyldiyl or heteroalkyldiyl bridge having from 2 to 5 backbone atoms such that the substituents and the nitrogen atom taken together form a ring structure, which may be saturated or unsaturated, but is preferably saturated. The bridge substituent may be branched or straight-chain, but is preferably straight-chain, e.g., ethano, propano, butano, etc. The ring structure may contain, in addition to the nitrogen atom of the aminopyridinium, one or more heteroatoms, which are typically selected from the group consisting of O, S and N. When the nitrogen atom is not included in a ring structure, the amino group is preferably dimethylamino. When the nitrogen atom is included in a ring structure, the ring is preferably a morpholino or piperazine ring. Particularly preferred Pyr+ groups are 4-(dimethylamino)pyridinium, 4-(morpholino) pyridinium, and 1-methyl-4-piperazinylpyridinium.
The alkylthio, arylthio or heteroarylthio group is attached to the phenyl ring via the sulfur atom and may also be substituted with one or more of the same or different substituents. The nature of the substituents will depend upon whether the group is an alkylthio, arylthio or heteroarylthio. The alkyl chain of an alkylthio group may be substituted with virtually any substituent, including, but not limited to, xe2x80x94X, xe2x80x94R, xe2x95x90O, xe2x80x94OR, xe2x80x94SR, xe2x95x90S, xe2x80x94NRR, xe2x95x90NR, xe2x80x94CX3, xe2x80x94CN, xe2x80x94OCN, xe2x80x94SCN, xe2x80x94NCO, xe2x80x94NCS, xe2x80x94NO, xe2x80x94NO2, xe2x95x90N2, xe2x80x94N3, xe2x80x94S(O)2Oxe2x88x92, xe2x80x94S(O)2OH, xe2x80x94S(O)2R, xe2x80x94C(O)R, xe2x80x94C(O)X, xe2x80x94C(S)R, xe2x80x94C(S)X, xe2x80x94C(O)OR, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)OR, xe2x80x94C(O)SR, xe2x80x94C(S)SR, xe2x80x94C(O)NRR, xe2x80x94C(S)NRR and xe2x80x94C(NR)NRR, where each X is independently a halogen (preferably xe2x80x94F or xe2x80x94Cl) and each R is independently hydrogen, (C1-C6) alkyl or heteroalkyl, (C5-C14) aryl or heteroaryl. The R groups may be further substituted with one or more of the same or different substituents, which are typically selected from the group consisting of xe2x80x94X, xe2x80x94Rxe2x80x2, xe2x95x90O, xe2x80x94ORxe2x80x2, xe2x80x94SRxe2x80x2, xe2x95x90S, xe2x80x94NRxe2x80x2Rxe2x80x2, xe2x95x90NRxe2x80x2, xe2x80x94CX3, xe2x80x94CN, xe2x80x94OCN, xe2x80x94SCN, xe2x80x94NCO, xe2x80x94NCS, xe2x80x94NO, xe2x80x94NO2, xe2x95x90N2, xe2x80x94N3, xe2x80x94S(O)2Oxe2x88x92, xe2x80x94S(O)2OH, xe2x80x94S(O)2Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94C(O)X, xe2x80x94C(S)Rxe2x80x2, xe2x80x94C(S)X, xe2x80x94C(O)ORxe2x80x2, xe2x80x94C(O)Oxe2x88x92, xe2x80x94C(S)ORxe2x80x2, xe2x80x94C(O)SRxe2x80x2, xe2x80x94C(S)SRxe2x80x2, xe2x80x94C(O)NRxe2x80x2Rxe2x80x2, xe2x80x94C(S)NRxe2x80x2Rxe2x80x2 and xe2x80x94C(NR)NRxe2x80x2Rxe2x80x2, where each X is independently a halogen (preferably xe2x80x94F or xe2x80x94Cl) and each Rxe2x80x2 is independently hydrogen, (C1-C6) alkyl or heteroalkyl, (C5-C14) aryl or heteroaryl.
Due to synthetic constraints, when the group is an arylthio or heteroarylthio, the aryl or heteroaryl rings should not be directly substituted with halogens, although halogens may be included in the substituent (e.g., a haloalkyl). Thus, when the group is an arylthio or a heteroarylthio, typical substituents include any of the above-listed alkylthio substituents, with the exception of halogen.
The rhodamine dyes of the invention may include a linker L that can be used to conjugate the dyes, preferably by way of covalent attachment, to other compounds or substances, such as peptides, proteins, antibodies, nucleoside/tides, polynucleotides, polymers, particles, etc. The identity of linker L will depend upon the nature of the desired conjugation. For example, the conjugation may be: (i) mediated by ionic interactions, in which case linker L is a charged group; (ii) mediated by hydrophobic interactions, in which case L is a hydrophobic moiety; (iii) mediated by covalent attachment, in which case L is a reactive functional group (Rx) that is either capable of forming a covalent linkage with another complementary functional group (Fx) or is capable of being activated so as to form a covalent linkage with complementary functional group Fx; or (iv) mediated through the use of pairs of specific binding molecules, such as biotin and avidin/streptavidin, in which case linker L is one member of the pair, e.g., biotin.
Linker L is attached to the rhodamine dyes of the invention at the rhodamine-type parent xanthene ring and/or it is included as a substituent on the alkylthio, arylthio or heteroarylthio group substituting the fully substituted phenyl ring. When linker L is attached to the rhodamine-type parent xanthene ring, it is typically attached to a xanthene nitrogen or at the xanthene C4 carbon. The rhodamine dyes may have multiple linking moieties, but preferably have only a single linking moiety.
Depending upon the particular application, linker L may be attached directly to the rhodamine dye, or indirectly through one or more intervening atoms that serve as a spacer. Linker L can be hydrophobic or hydrophilic, long or short, rigid, semirigid or flexible, depending upon the particular application. When L is positioned at the alkylthio, arylthio or heteroarylthio group, it is preferably attached directly to the molecule. In this latter embodiment, L is a bond.
The new, fully substituted phenyl rings described herein can be used to replace the xe2x80x9cbottom ringxe2x80x9d or xe2x80x9cbottom substituent,xe2x80x9d i.e., the substituent attached to the xanthene C9 carbon, of virtually any rhodamine dye that is known in the art or that will be later developed. Thus, the new, fully substituted phenyl rings described herein can be covalently attached to the C-9 position of virtually any rhodamine-type parent xanthene ring that is now known or that will be later developed to yield a rhodamine dye without longer absorption and emission maxima and with greater water-solubility. As the new bottom rings do not deleteriously affect the photostability properties that are characteristic of rhodamine dyes, the new dyes are also highly photostable. Exemplary rhodamine-type parent xanthene rings that can comprise the rhodamine dyes of the invention include, by way of example and not limitation, the xanthene rings (xe2x80x9ctop ringsxe2x80x9d) of the rhodamine dyes described in U.S. Pat. No. 5,936,087; U.S. Pat. No.5,750,409; U.S. Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,840,999; U.S. Pat. No. 5,847,162; U.S. application Ser. No. 09/038,191, filed Mar. 10, 1998; U.S. application Ser. No. 09/277,793, filed Mar. 27,1999; U.S. application Ser. No. 09/325,243, filed Jun. 3, 1999; PCT Publication WO 97/36960; PCT Publication WO 99/27020; Sauer et al., 1995, J. Fluorescence 5(3):247-261; Arden-Jacob, 1993, Neue Lanwellige Xanthen-Farbstoffe fxc3xcr Fluoreszenzsonden und Farbstoff Laser, Verlag Shaker, Germany; and Lee et al., 1992, Nucl. Acids Res. 20(10):2471-2483. Preferred rhodamine-type parent xanthene rings are fluorescent.
In another aspect, the invention provides labeled conjugates comprising a rhodamine dye according to the invention and another molecule or substance. The rhodamine dye is conjugated to the other molecule or substance, typically via covalent attachment, through linker L, as previously described. Once conjugated, the rhodamine dye provides a convenient fluorescent label for subsequent detection. The rhodamine dyes of the invention can be used to fluorescently label a wide variety of molecules and substances, including but not limited to, amino acids, peptides, proteins, antibodies, enzymes, receptors, nucleosides/tides, nucleoside/tide analogs, polynucleotides, polynucleotide analogs, nucleic acids, carbohydrates, lipids, steroids, hormones, vitamins, drugs, metabolites, toxins, organic polymers, etc. The dyes can also be used to label particles such as solid phase synthesis substrates, nanoparticles, microspheres or liposomes. In embodiments involving nanoparticles, microspheres and/or liposomes, the dye need not include a linking moiety. It can be incorporated into the various particles during their formation. The molecule or substance may be labeled with one or more rhodamine dyes of the invention, which may be the same or different.
In one embodiment, a rhodamine dye of the invention is covalently conjugated to another dye compound to form an energy-transfer dye pair. The energy-transfer dye pair can be further conjugated to other molecules or substances, as described above, to provide an energy-transfer label. The energy-transfer dye pair generally comprises a donor dye (DD), an acceptor dye (AD), and a linkage linking the donor and acceptor dyes. The donor dye is capable of absorbing light at a first wavelength and emitting excitation energy in response. The acceptor dye is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto. While in many instances the emission wavelength of the donor dye and the excitation wavelength of the acceptor dye will overlap, such overlap is not required. The acceptor dye need only fluoresce in response to the donor dye absorbing light, regardless of the mechanism of action. The linkage serves to facilitate efficient energy transfer between the donor and acceptor dyes. According to this aspect of the invention, at least one of the donor and acceptor dyes is a rhodamine dye according to the invention. Preferably, the acceptor dye is a rhodamine dye according to the invention and the donor dye is a xanthene dye, most typically a fluorescein dye. The exact nature or identity of the donor dye will depend upon the excitation and emission properties of the rhodamine acceptor dye, and will be apparent to those having skill in the art. When covalently conjugated to enzymatically-incorporatable nucleotides, such as dideoxynucleotide 5xe2x80x2-triphosphates, i.e. xe2x80x9cterminatorsxe2x80x9d, such energy-transfer dyes are ideally suited for use in sequencing nucleic acids.
Since the rhodamine dyes of the invention may comprise virtually any rhodamine-type parent xanthene ring, the dyes cover a broad range of the visible spectrum, ranging from green to red. Thus, both dyes of an energy-transfer dye pair may be rhodamine dyes of the invention. In this embodiment, one dye of the invention acts as the donor and another as the acceptor, depending upon their spectral properties.
In another embodiment, a rhodamine dye of the invention, or an energy-transfer dye pair including a rhodamine dye of the invention, is covalently conjugated to a nucleoside/tide, nucleoside/tide analog, polynucleotide or polynucleotide analog to form a labeled conjugate therewith. The dye or dye pair is typically conjugated to the nucleobase moiety of the respective nucleoside/tide, polynucleotide or analog, but may be conjugated to other portions of the molecule, such as the 5xe2x80x2-terminus, 3xe2x80x2-terminus and/or the phosphate ester intenucleoside linkage.
In one preferred embodiment, the labeled conjugate is a labeled polynucleotide or polynucleotide analog that can be used as a primer for generating labeled primer extensions products via template-directed enzymatic synthesis reactions. In another preferred embodiment, the labeled conjugate is a labeled terminator. When used in conjunction with enzymatically-extendable nucleotides or nucleotide or analogs, appropriate polymerizing enzymes and a primed template nucleic acid, such labeled terminators can be used to generate a series of labeled primer extension products via template-directed enzymatic synthesis for applications such as nucleic acid sequencing.
In a final aspect, the invention provides methods of using the rhodamine dyes or energy-transfer dye pairs of the invention to sequence a target nucleic acid. The method generally comprises forming a series of differently-sized primer extension products labeled with a rhodamine dye or energy-transfer dye pair of the invention, separating the series of differently-sized labeled extension products, typically based on size, and detecting the separated labeled extension products based on the fluorescence of the label. The sequence of the target nucleic acid is then assembled according to known techniques.
The series of differently-sized labeled extension products can be conveniently generated by enzymatically extending a primer-target hybrid according to well-known methods. For example, the series of labeled extension products can be obtained using a primer labeled with a rhodamine dye or dye pair of the invention and enzymatically extending the labeled primer-target hybrid in the presence of a polymerase, a mixture of enzymatically-extendable nucleotides or nucleotide analogs capable of supporting continuous primer extension (e.g., dATP, dGTP, dCTP and dUTP or dTTP) and at least one, typically unlabeled, terminator that terminates primer extension upon incorporation (e.g., a ddNTP). Alternatively, the series of labeled extension products can be obtained using an unlabeled primer and enzymatically extending the unlabeled primer-target hybrid in the presence of a polymerase, a mixture of enzymatically-extendable nucleotides or nucleotide analogs capable of supporting continuous primer extension and at least one terminator labeled with a rhodamine dye or energy-transfer dye pair of the invention. In either embodiment, the polymerase serves to extend the primer with enzymatically-extendable nucleotides or nucleotide analogs until a terminator is incorporated, which terminates the extension reaction. Once terminated, the series of labeled extension products are separated, typically based on size, and the separated labeled extension products detected based on the fluorescence of the labels.
In a particularly advantageous embodiment of this method, a mixture of four different terminators are used in a single extension reaction. Each different terminator is capable of terminating primer extension at a different template nucleotide, e.g., a mixture of 7-deaza-ddATP, ddCTP, 7-deaza-ddGTP and ddTTP or ddUTP, and is labeled with a different, spectrally-resolvable fluorophore, at least one of which is a rhodamine dye or energy-transfer dye pair according to the invention. According to this embodiment, an unlabeled primer-target nucleic acid hybrid is enzymatically extended in the presence of a polymerase, a mixture of enzymatically-extendable nucleotides or nucleotide analogs capable of supporting continuous primer extension and a mixture of the four different labeled terminators. Following separation based on size, a series of separated labeled extension products is obtained in which the emission properties (i.e., color) of each separated extension product reveals the identity of its 3xe2x80x2-terminal nucleotide. In a particularly preferred embodiment, all of the labeled terminators are excitable using a single light source.
Alternatively, terminators may be used in the absence of enzymatically-extendable nucleotides. In this instance, the primer is extended by only a single base. Again, the primer may be labeled, or, alternatively, one or more of the terminators may be labeled. Preferably, a mixture of four different labeled terminators is used, as described above. These xe2x80x9cmini sequencingxe2x80x9d embodiments are particularly useful for identifying polymorphisms in chromosomal DNA or cDNA.